This invention relates to newly identified polynucleotides, polypeptides encoded by such polynucleotides, the use of such polynucleotides and polypeptides, as well as the production of such polynucleotides and polypeptides. More particularly, the polypeptide of the present invention has been putatively identified as human cystatin E, sometimes hereinafter referred to as xe2x80x9cCysExe2x80x9d. The invention also relates to inhibiting the action of such polypeptides.
The cystatin superfamily comprises a group of cysteine proteinase inhibitors which are widely distributed in human tissues and body fluids, and which form tight and reversible complexes with cysteine proteinases such as cathepsins B, H, L, and S. The cystatins are most likely involved in the regulation of normal or pathological processes in which these proteinases participate. Thus, cystatins may influence the intra- and extracellular catabolism of proteins and peptides (Barret, A. J. and Kirchke, H., Methods Enzymol., 80:535-561 (1981)), regulate proteolytic processing of pro-hormones (Orlowski, M., Mol. Cell. Biochem., 52:49-74 (1983)) and pro-enzymes (Taugner, R., et al., Histochemistry, 83:103-108 (1985)), protect against penetration of normal tissues by malignant cells (Sloane, B. F., Semin. Cancer Biol., 1:137-152 (1990)) or microorganisms (Bjorck, L., et al., Nature, 337:385-386 (1989) and Bjorck, L., et al., J. Virol., 64:941-943 (1990)) and modulate local inflammatory processes in rheumatoid arthritis (Mort, J. S., et al., Arthritis Rheum., 27:509-515 (1984)) and purulent bronchiectasis (Buttle, D. J., et al., Scand. J. Clin. Lab. Invest., 50:509-516 (1990)).
The cystatin superfamily has been sub-divided into families I, II and III (also called the stefin, cystatin and kininogen families, respectively), each with members differing from those of the other families in structural organization and biological distribution (Barret, A. J., et al., Biochem. J., 236:312 (1986)). The family I cystatins A and B are small proteins consisting of single polypeptide chains of about 100 amino acid residues without disulfide bridges. The family II cystatins consist of polypeptide chains of approximately 120 amino acid residues with two intra-chain disulfide bonds. Finally, the family III cystatins, the kininogens, display a higher degree of structural complexity characterized by the presence of three family II cystatin-like domains, each with two disulfide bridges at positions homologous to those in family II cystatins (Muller-Esterl, W., et al., Transbiochem. Sci., 11:336-339 (1986)). Family I and II cystatins are mainly present intracellularly and in secretory fluids (Abrahamson, M., et al., J. Biol. Chem., 261:11282-11289 (1986)), whereas kininogens are highly concentrated in blood plasma (Adam, A., et al., Clin. Chem., 31:423-426 (1985)).
At least one type II cystatin, designated cystatin C, appears to be expressed in all tissues (Abrahamson, M., et al., Biochem. J., 268:287-294 (1990)). In contrast, S-type cystatins are found predominantly in saliva (Abrahamson, M., et al., J. Biol. Chem., 261:11282-11289 (1986)). Cystatins and derivative peptides possess antibacterial and antiviral activities (Bjorck, et al. (1989, 1990)), consistent with their presence in secretions bathing epithelial surfaces directly exposed to the environment. The cystatins may also modulate the immune response. This could occur directly, by inhibiting cysteine protease releases by macrophages (Bieth, J., Cysteine Proteinases and Their Inhibitors, V. Turk, ed. (Walter De Gruyter and Company, New York) pp. 693-703 (1986)), or indirectly, by inhibiting the chemotaxic response and the phagocytosis-associated respiratory burst of the cells (Leung-Tack, et al., Biol. Chem., 371:255-258 (1990)). This data suggests that type II cystatins might perform a variety of protective functions at epithelial surfaces. The human type II cystatin gene family consists of at least seven members.
The disease hereditary cystatin C amyloid angiopathy (HCCAA) is associated with a Glu to Leu mutation in the gene encoding cystatin C. This leads to deposition of amyloid fibrils comprised of this mutant cystatin C in the cerebral arteries, which appears to cause fatal hemorrhaging (Ghiso, J., et al., PNAS, USA, 83:2974-2978 (1986)).
The polypeptide of the present invention has been putatively identified as a CysE as a result of amino acid sequence homology to cystatin C and on conservation of cystatin-like functional motifs in its amino acid sequence.
The present invention provides isolated nucleic acid molecules comprising a polynucleotide encoding the CysE polypeptide having the amino acid sequence shown in FIG. 1 (SEQ ID NO:2) or the amino acid sequence encoded by the cDNA clone deposited with the American Type Culture Collection (ATCC(copyright), located at 10801 University Boulevard, Manassas, Va. 20120-2209, USA) in a bacterial host as ATCC(copyright) Deposit Number 97156 on May 22, 1995. The nucleotide sequence determined by sequencing the deposited CysE clone, which is shown in FIG. 1 (SEQ ID NO:1), contains an open reading frame encoding a polypeptide of 149 amino acid residues, with a leader sequence of about 28 amino acid residues, and a predicted molecular weight of about 14 kDa. The amino acid sequence of the mature CysE protein is shown in FIG. 1, amino acid residues 29-149 (SEQ ID NO:2).
Thus, one aspect of the invention provides an isolated nucleic acid molecule comprising a polynucleotide having a nucleotide sequence selected from the group consisting of: (a) a nucleotide sequence encoding the CysE polypeptide having the complete amino acid sequence in FIG. 1 (SEQ ID NO:2); (b) a nucleotide sequence encoding the mature CysE polypeptide having the amino acid sequence at positions 29-149 in FIG. 1 (SEQ ID NO:2); (c) a nucleotide sequence encoding the CysE polypeptide having the complete amino acid sequence encoded by the cDNA clone contained in ATCC(copyright) Deposit No. 97156; (d) a nucleotide sequence encoding the mature CysE polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC(copyright) Deposit No. 97156; and (e) a nucleotide sequence complementary to any of the nucleotide sequences in (a), (b), (c) or (d) above.
Further embodiments of the invention include isolated nucleic acid molecules that comprise a polynucleotide having a nucleotide sequence at least 90% identical, and more preferably at least 95%, 96%, 97%, 98% or 99% identical, to any of the nucleotide sequences in (a), (b), (c), (d) or (e), above, or a polynucleotide which hybridizes under stringent hybridization conditions to a polynucleotide in (a), (b), (c), (d) or (e), above. This polynucleotide which hybridizes does not hybridize under stringent hybridization conditions to a polynucleotide having a nucleotide sequence consisting of only A residues or of only T residues. An additional nucleic acid embodiment of the invention relates to an isolated nucleic acid molecule comprising a polynucleotide which encodes the amino acid sequence of an epitope-bearing portion of a CysE polypeptide having an amino acid sequence in (a), (b), (c) or (d), above.
The present invention also relates to recombinant vectors, which include the isolated nucleic acid molecules of the present invention, and to host cells containing the recombinant vectors, as well as to methods of making such vectors and host cells and for using them for production of CysE polypeptides or peptides by recombinant techniques.
The invention further provides an isolated CysE polypeptide having an amino acid sequence selected from the group consisting of: (a) the amino acid sequence of the CysE polypeptide having the complete 149 amino acid sequence, including the leader sequence shown in FIG. 1 (SEQ ID NO:2); (b) the amino acid sequence of the mature CysE polypeptide (without the leader) having the amino acid sequence at positions 29-149 in FIG. 1 (SEQ ID NO:2); (c) the amino acid sequence of the CysE polypeptide having the complete amino acid sequence, including the leader, encoded by the cDNA clone contained in ATCC(copyright) Deposit No. 97156; and (d) the amino acid sequence of the mature CysE polypeptide having the amino acid sequence encoded by the cDNA clone contained in ATCC(copyright) Deposit No. 97156. The polypeptides of the present invention also include polypeptides having an amino acid sequence with at least 90% similarity, and more preferably at least 95% similarity to those described in (a), (b), (c) or (d) above, as well as polypeptides having an amino acid sequence at least 80% identical, more preferably at least 90% identical, and still more preferably 95%, 96%, 97%, 98% or 99% identical to those above.
An additional embodiment of this aspect of the invention relates to a peptide or polypeptide which has the amino acid sequence of an epitope-bearing portion of a CysE polypeptide having an amino acid sequence described in (a), (b), (c) or (d), above. Peptides or polypeptides having the amino acid sequence of an epitope-bearing portion of a CysE polypeptide of the invention include portions of such polypeptides with at least six or seven, preferably at least nine, and more preferably at least about 30 amino acids to about 50 amino acids, although epitope-bearing polypeptides of any length up to and including the entire amino acid sequence of a polypeptide of the invention described above also are included in the invention. In another embodiment, the invention provides an isolated antibody that binds specifically to a CysE polypeptide having an amino acid sequence described in (a), (b), (c) or (d) above.
The invention further provides methods for isolating antibodies that bind specifically to a CysE polypeptide having an amino acid sequence as described herein. Such antibodies are useful diagnostically or therapeutically as described below.
The present invention also provides a screening method for identifying compounds capable of enhancing or inhibiting modulation of proteinase activity by CysE, which involves contacting CysE with a proteinase in the presence of the candidate compound, assaying the ability of the proteinase to cleave a substrate in the presence of CysE and the candidate compound, and comparing the result with a standard, the standard being assayed when contact is made in absence of the candidate compound; whereby, an increased in substrate cleavage over the standard indicates that the compound is an agonist and a decreased substrate cleavage over the standard indicates that the compound is an antagonist.
In another aspect, a screening assay for agonists and antagonists is provided which involves determining the effect a candidate compound has on CysE binding to a CysE binding molecule. In particular, the method involves contacting a CysE binding molecule with a CysE polypeptide and a candidate compound and determining whether CysE polypeptide binding to the CysE binding molecule is increased or decreased due to the presence of the candidate compound.
The present inventors have discovered that CysE is expressed in amniotic cell, fetal skin and placental tissues. For a number of disorders related to fetal development, it is believed that significantly higher or lower levels of CysE gene expression can be detected in affected tissues taken from an individual having, or carrying a child having, such a disorder, relative to a xe2x80x9cstandardxe2x80x9d CysE gene expression level, i.e., the CysE expression level in healthy tissue from an individual not having, or carrying a child not having the fetal development disorder. Thus, the invention provides a diagnostic method useful during diagnosis of disorders, such as fetal development disorders, which involves: (a) assaying CysE gene expression level in cells or body fluid of an individual; (b) comparing the CysE gene expression level with a standard CysE gene expression level, whereby an increase or decrease in the assayed CysE gene expression level compared to the standard expression level is indicative of the disorder.
An additional aspect of the invention is related to a method for treating an individual in need of an increased level of CysE activity in the body comprising administering to such an individual a composition comprising a therapeutically effective amount of an isolated CysE polypeptide of the invention or an agonist thereof.
A still further aspect of the invention is related to a method for treating an individual in need of a decreased level of CysE activity in the body comprising, administering to such an individual a composition comprising a therapeutically effective amount of a CysE antagonist. Preferred antagonists for use in the present invention are CysE-specific antibodies.